Recent advances suggest that self-replication of protein physical states directs both the development and spread of the Transmissible Spongiform Encephalopathies and the inheritance of some phenotypic traits in lower eukaryotes. This novel biological process, known as the prion hypothesis, predicts that a unique group of proteins has the capacity to adopt multiple conformational states with distinct physiological consequences in vivo. Since one-fold-one-function proteins are unable to act in roles that have historically been linked to nucleic acids such as infectivity and inheritance, understanding how a prion protein's structure can be constrained to allow the faithful propagation of associated phenotypes but remain sufficiently flexible to allow occasional transitions in state is crucial to understanding the physiological consequences of the protein- only hypothesis. The prion cycles of lower eukaryotes provide experimentally tractable model systems for studying prion cycle regulation in vivo. For example, the Sup35 protein of S. cerevisiae is a component of the translation termination complex whose function is reversibly modulated by a prion cycle. In the non-prion state, Sup35 facilitates efficient termination (psi- phenotype), but in the prion form, Sup35's activity is compromised leading to stop codon read-through ([PSI+] phenotype). While the [PSI+] and [psi-] phenotypes are largely stable, they spontaneous interconvert (about 1 cell/million) and can be induced to quantitatively switch by chemical and molecular stimuli. Using this system, we will begin to elucidate the molecular mechanism underlying the near-faithful propagation of prion forms in vivo by focusing on two contributing factors: the interplay of distinct forms when present in the same cell and the trans regulators of efficient prion conversion. Toward this end, we will 1) determine the molecular basis of prion variant dominance in vivo, 2) elucidate the molecular mechanisms by which known regulators of the Sup35/[PSI+] prion cycle modulate propagation and phenotypic transitions, and 3) screen for and characterize novel prion regulators. Together, these lines of investigation will build a framework for understanding protein-only phenotypic propagation in terms of prion protein biogenesis. A strong foundation of previous work indicates that the knowledge gleaned from prion studies in lower eukaryotes is clearly and directly applicable to our understanding of prion mechanisms and their physiological consequences in mammals.